The Perkin-Elmer Corporation

Pe Corporation

Applied Biosystems Division, Perkin-Elmer Corp.

Susan J Flood

Kenneth J Livak

Khairuzzaman Bashar Mullah

Jeffrey M Marmaro

Jeffrey Mamoro

A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

Hybridization assay using self-quenching fluorescence probe

An oligonucleotide probe is provided which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

Self-quenching fluorescence probe

A method is provided for monitoring the progress of nucleic acid amplifications that rely on a nucleic acid polymerase having 5'.fwdarw.3' exonuclease activity. An important feature of the method is providing an oligonucleotide probe having a reporter molecule and a quencher molecule at either end such that the quencher molecule substantially quenches any fluorescence from the reporter whenever the oligonucleotide probe is in a single stranded state and such that the reporter is substantially unquenched whenever the oligonucleotide probe is in a double stranded state hybridized to a target polynucleotide.

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Fremont, CA, United States of America

Susan J Flood